1. Field of the Invention
The invention relates to a process for extracting and purifying a protein having an apparent molecular weight of about 69,000 daltons from the outer membrane of the bacterium Bordetella pertussis. The protein so extracted and purified may be utilized as a component of an acellular pertussis vaccine.
2. Description of the Prior Art
The bacterium Bordetella pertussis is the causative agent of the serious infectious disease known as pertussis or whooping cough. Vaccines which are used to immunize infants and children are composed of whole cells of B. pertussis inactivated with chemical agents or heat. While whole cell vaccines contain the antigenic components necessary to elicit protective immunity, they may also contain substances irrelevant to protection and possibly related to undesirable side effects of immunization.
In an effort to minimize any undesirable side effects of immunization with whole cell vaccines, the pathogenic mechanisms of B. pertussis have been studied to determine which antigenic components contribute to protective immunity. The specific antigens so identified thus could be included in an acellular vaccine which would confer immunity to disease without introducing irrelevant and possibly undesirable substances in the immunization. A number of antigens have been proposed as acellular vaccine components, for example: lymphocytosis promoting factor (LPF; also known as histamine sensitizing factor, islet activating protein, and pertussis toxin), filamentous hemagglutinin (FHA), and fimbrial agglutinogens. (Weiss, A.A. and Hewlett, E.L., 1986, Ann. Rev. Microbiol., 40:661-686.)
Another antigen, associated with the B. pertussis outer membrane, and having an apparent molecular weight of about 69,000 daltons is referred to as the B. pertussis 69K protein or P.69. The B. pertussis 69K protein is immunologically related to similar proteins, having slight differences in electrophoretic mobility, produced by the human pathogen B. parapertussis and the animal pathogen B. bronchiseptica. It has been suggested that the 69K protein would be a protective antigen useful as a vaccine component. The correlation between protection against disease and the presence of specific antibodies was first established for the B. bronchiseptica 68K protein, and the investigation was later extended to the 69K protein of B. pertussis by Novotny et al. (Novotny, P., Chubb, A. P., Cownley, K., Montaraz, J. A., and Beesley, J. E., 1985. Dev. Biol. Stand. 61: 27-41; Novotny, P., Korisch, M., Cownley, K., Chubb, A. P., and Montaraz, J. A., 1985. Infect. Immun. 50: 190-198; Montaraz, J. A., Novotny, P., and Ivanyi J., 1985. Infect. Immun. 47: 744-751; and Novotny, P., Chubb, A. P., Cownley, K., and Montaraz, J. A., 1985. Infect. Immun. 50: 199-206). Others have shown that 69K is protective in certain animal models of pertussis, either by active or passive immunity. Furthermore, antibodies against 69K are present in sera of humans who have recovered from pertussis. The 69K protein has the properties of a B. pertussis agglutinogen, and can act as an adhesin, causing attachment to mammalian cells. The 69K protein is expressed coordinately with LPF, FHA, and other specific factors under the control of a genetic locus related to B. pertussis virulence, and is expressed only in virulent strains. Finally, the 69K protein has been reported to be a component of an acellular pertussis vaccine successfully utilized in Japan. (Aoyama, J., Murase, Y., Kato, M., Iwai, H., and Iwata, J., 1989, Am. J. Dis. Child. 143: 655-659; Shahin, R. D., Brennan, M. J., Li., Z. M., Meade, B. D., and Manclark, C. R., 1989, J. Exp. Med. (in press); Brennan, M. J., Li, Z. M., Lowell, J. L., Bisher, M. E., Steven, A. C., Novotny, P., and Manclark, C. R., 1988. Infect. Immun. 56: 3189-3195; Charles, I. G., Dougan, G., Pickard, D., Chatfield, S., Smith, M., Novotny, P., Morrissey, P., and Fairweather, N. F., 1989. Proc. Natl. Acad. Sci. USA 86: 3554-3558; Leininger - Zapata, E., Brennan, M. J., Kenimer, J. G., Charles, I., Fairweather, N., and Novotny, P. 1989, Abstr. Amer. Microbiol. p.51, abstr. B-123; and Shahin, R., Brennan, M. J., and Meade, B. D., 1989. Abstr. Amer. Soc. Microbiol. p.51, abstr. B-125.)
In order to employ the 69K protein as a component of an acellular pertussis vaccine, a method is required for the efficient purification of the protein applicable to commercial production scale. Previously described methods, however, have several disadvantages for adaptation to large scale.
Novotny et al (Infection and Immunity 50: 199-206 (1985); EP 0 162 639) describe a method for extracting and purifying 69K protein from whole B. pertussis cells. The method described by Novotny et al includes suspending the whole B. pertussis cells in water, adjusting to pH3 with a buffer, incubating at 37.degree. C. for approx 18 hours to release proteins including the 69K protein from the whole cells, removing the cells by centrifugation leaving a protein extract, precipitating the protein extract with acetone at -20.degree. C., and centrifuging the precipitate to separate the precipitated proteins from non-proteinaceous material remaining in solution. The resulting protein extract was chromatographed on a DEAE - Trisacyl ion-exchange column and eluted a with salt gradient. Material not retained by the ion-exchange column was subjected to isoelectric focusing with a preparative isoelectic focusing gel or chromatofocusing. Protein containing fractions as recognized by an anti-69K protein monoclonal antibody were then applied to a monoclonal antibody column which was then eluted with 6M urea to produce the 69K protein.
This method would not be conducive to commercial production of the 69K protein, however, because it requires 18 hours for extraction of the protein from cells, utilizes acetone, a dangerous solvent, in the precipitation step, requires an ion-exchange chromatography column run lasting 20 hours, and utilizes a cumbersome preparative isoelectric focusing procedure lasting 16 hours. Further, the resulting protein preparation is not completely purified until after an additional step where it is passed through an affinity column utilizing anti-69K monoclonal antibodies linked to a polymeric support and eluted utilizing a harsh 6M urea. Finally, the protein is unstable throughout the purification process resulting in some degradation of the protein during purification.
Brennan et al [Infection and Immunity 56: 3189-3195 (1988)] also describe a method for extracting and purifying the B. pertussis 69K protein from whole B. pertussis cells. The method described by Brennan et al includes suspending live B. pertussis cells in a phosphate buffered saline solution, incubating at 60.degree. C. for 1 hour to release the proteins from the cells, removing the cells by centrifugation leaving a protein extract and dialyzing the protein extract so obtained in a tris-buffered saline solution which contains the protease inhibitors ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonylfluoride (PMSF), and Brij 35, a detergent. The extract is then applied to a fetuin-Sepharose column to remove pertussis toxin, followed by a monoclonal antibody affinity column, which is eluted with 6M urea to produce the 69K protein. As with the method disclosed by Novotny et al, this method is subject to some limitations which make it relatively difficult to carry out efficiently on a commercial scale. For example, comparing the multiple incubation extraction steps of the present invention to the single step of Brennan et al demonstrates that the single incubation extraction step results in a very incomplete extraction of 69K protein from the cells. Further, the method disclosed by Brennan et al would require the large scale use of toxic and expensive chemicals if it were practiced on a commercial scale. Finally, both the Novotny et al and Brennan et al methods require the use of a monoclonal antibody affinity column.
In general, methods employing monoclonal antibody affinity columns are not suited to commercial production because the antibodies are not commercially available and must be generated in a lengthy procedure of immunization followed by hybridoma production and screening. The antibodies must then be purified and linked to the affinity support, all of which is both labor and time consuming. Furthermore, elution from the affinity columns involves high concentrations of urea which is detrimental because urea is a known denaturing agent and can alter protein structural characteristics.
A third method for extracting and purifying 69K protein from whole B. pertussis cells is described by Burns et al in U.S. Ser. No. 7/308,864 filed Feb. 10, 1989, now U.S. Pat. No. 5,101,014, and in abstracts of American Society of Microbiology, p. 51, abstract B-126 (1989). This method includes suspending live B. pertussis cells in a tris-buffered saline solution which contains PMSF and sodium azide, incubating at 60.degree. C. for 1 hour to release proteins from the cells, removing the cells by centrifugation leaving a protein extract, and dialyzing the protein extract so obtained in a tris-buffered saline solution which contains the protease inhibitor, PMSF, at substantial concentration (1 mM). The extract is then applied to a DEAE - sepharose ion-exchange column which is eluted with a salt gradient. The eluate is dialyzed and then passed to a dye ligand chromatography column (Affi-gel Blue (Bio-Rad)) and eluted with a high concentration of urea in the presence of a detergent to produce the 69K protein. As with the Novotny et al and Brennan et al methods, this method is also not suitable to commercial production of the 69K protein because it requires the presence of protease inhibitors at substantial concentration (e.g. 1 mM PMSF) throughout the process to preserve the 69K protein from proteolytic degradation. For adaptation to large scale, the use of large volumes of buffers containing such toxic and expensive substances is a disadvantage, and the complete removal of these substances from the final product must be assured. In addition, the urea and detergent materials used to elute the protein from the dye ligand column may adversely affect the protein structure via denaturation.
Accordingly, it is a object of the present invention to provide an improved process for the extraction and purification of 69K protein from whole B. pertussis cells, which process is suitable to commercial production of the protein. It is another object to provide a process in which the yield of protein is increased. It is another object to provide a process in which the stability of the 69K protein is increased. It is a further object to provide a process in which toxic protease inhibitors or harsh denaturing agents such as a high concentration of urea are not employed.